Synaptic transmission is key for interneuronal communication and is mediated by neurotransmitters that are released by synaptic vesicle exocytosis. Insights into the mechanisms of neurotransmitter release and its regulation are essential for understanding how the brain processes information, and how synaptic transmission is affected in diseases such as Parkinson's disease, Alzheimer's disease, and drug addiction. This research is also relevant to cell biology in general because of its importance to understand intracellular membrane fusion, and to other diseases that arise from defects of regulated secretion, which plays a wide variety of physiological functions such as control of heart rate, blood pressure or insulin release. Release is governed by a complex protein machinery that includes proteins with homologues in most types of intracellular membrane fusion such as the SNARE proteins and munc18-1. In addition, several proteins such as munc13-1 and complexins play critical roles that are specialized for the tight spatial and temporal regulatory requirements of neurotransmitter release. While the research performed under this grant and studies from other laboratories have yielded key insights into how these proteins function, the mechanism of release is still unclear and fundamental questions remain unanswered. Our research has recently identified multiple weak interactions between the components of the release machinery that could play critical roles because they can catalyze structural rearrangements and can be enhanced by cooperativity. Elucidating the structural basis for these interactions is essential to take a quantum leap and gain a true understanding of the mechanism of release. Moreover, the influence of membranes on these interactions needs to be characterized, ideally in the context of trans-SNARE complexes bridging two apposed membranes. The research proposed in this application is designed to address these questions and will test emerging models for the functions of these proteins, with the goal of developing a detailed picture of the mechanism of release that integrates all their functions. This research involves an interdisciplinary approach integrating structural studies at atomic resolution, biochemical experiments, reconstitution assays and electrophysiological analyses of neurotransmitter release in neurons performed by collaborators. Four specific aims are proposed that focus on: 1. Munc18-1-SNARE interactions; 2. Munc13-1 function in fusion; 3. Dual roles of complexin-1; and 4. Reconstituting basic steps of neurotransmitter release. The results of this research will not only be important to understand the mechanisms of neurotransmitter release and membrane fusion in general, but will also have an impact in the design of therapies for the diverse diseases mentioned above.